Only a fraction of the more than 20,000 genes in the nematode C. elegans have been amenable to traditional methods of genetic study. In yeast systematic investigations of double mutants, exploiting the yeast deletion collection, have revealed interactions between genes across the genome and have permitted inference of function for most genes of otherwise unknown function. Here we propose to develop methods that will permit analogous studies in C. elegans. We will exploit a recently completed resource of sequenced mutant strains that contains nonsynonymous changes in almost every gene in the genome. These strains will be exposed to RNAi to look for interactions as reflected in differences compared to the RNAi against wild type and other mutant strains or the strain in the absence of RNAi. To increase the efficiency and simplify comparison, the strains will be competed against one another in pools of 100 or more, using growth as a surrogate for a host of phenotypes. The abundance of individual strains in the pools will be followed using molecular inversion probes (MIPs) for the unique mutations quantified by high-throughput sequencing. By comparing the abundance relative to controls, other RNAs and population genetic models, we will determine which strains show a significant positive or negative interaction with each RNAi. To determine which of the various mutations within each strain underlie the interaction with the RNAi, we will cross each interacting strain with a multiply marked strain, compete the progeny in a pool with the RNAi and use bulk segregant analysis to identify the interacting loci. We will test the methods using a battery of RNAi's from genes that include positive and negative controls from the literature, from genes of the twk- family of potassium channels that act as dimers but are individually dispensable, and from genes broadly representative of the genome to assess the overall efficiency of the methods. The successful implementation of these methods will pave the way for systematic investigation of SGA across the worm genome, providing valuable insight into the role of genes without previously known function. It will also provide a framework for more detailed investigations of these genes and networks. It may also inspire the development of analogous methods for still more complex organisms. A more comprehensive understanding of genetic interactions in this model metazoan can in turn provide insight into likely gene-gene interactions in human.